This proposal addresses the general question of how developmental processes are regulated at the level of gene expression. The specific goal is to investigate the mechanisms by which alternative RNA processing controls the sex-specific expression of a gene that regulates somatic sexual differentiation in Drosophila melanogaster. The doublesex (dsx) gene acts as a "genetic switch"; it is expressed in either a male- or female-specific mode, the choice of which directs the course of somatic sexual development. The regulation of dsx occurs through sex-specific RNA processing. To understand how this regulation occurs, a three-stage strategy is proposed: (i) Identify and characterize the dsx sequences required in cis for sex- specific RNA splicing. This will be done by in vivo studies using dsx minigene constructs. The effect of site-directed mutations on the minigene splicing pattern will define the organization and behavior of these sites. (ii) Determine how these sites function, i.e., do they bind trans-acting regulatory factors and are they involved in important secondary structures? These studies will utilize RNA-protein binding assays including gel mobility shift and protection experiments. (iii) Isolate and characterize the trans-acting factors. The proposed research will contribute to our understanding of a developmental process that is fundamental to all sexually reproducing organisms. Furthermore, the insights gained on the regulation of RNA processing will have substantial relevance to the study of a number of genes that are regulated in a similar manner. These include genes of major medical importance involving such processes as oncogenesis, viral regulation, and the immune system, among others.